Field of the Invention
The invention generally relates to recombinant Mycobacteria which contain and express sequences encoding a heparin-binding hemagglutinin (HBHA) fusion protein. The fusion protein contains an amino terminal mycobacterial antigen Ag85B leader sequence, and transcription of the fusion protein is driven by a suitable promoter, e.g. the Ag85B promoter. The invention also provides methods of making and using the recombinant Mycobacteria and the recombinant fusion protein, e.g. as vaccinogens.
Background of the Invention
Tuberculosis (TB) is a global public health problem resulting in 8 million new cases and 2 million deaths each year. A particularly problematic aspect of TB diagnosis and treatment is the ability of the Mycobacterium tuberculosis (Mtb) bacillus to enter a latent, asymptomatic state and to persist in latently infected individuals for long periods of time. Such individuals are susceptible to reactivation of the disease due to, for example, immune suppression caused by diseases or conditions such as HIV, treatments such as chemotherapy and the use of corticosteroids, the waning of immunity that accompanies aging, etc. An estimated 2 billion persons (one-third of the world's population) are latently infected with Mtb at present, and activation of latent tuberculosis accounts for most new cases of active disease. Reactivation is associated with inflammation, necrosis and cavitation of the lung, a process that results in draining of the lesions into the bronchus. Aerosols generated when individuals with bronchial lesions cough causes dissemination of the Mtb organism to uninfected, susceptible persons, and the transmission cycle is thus maintained.
The only currently available vaccine against TB, Mycobacterium bovis (Bacille Calmette-Guérin) (BCG), was first introduced in 1921. BCG has been widely utilized and while studies show that for some purposes BCG is effective (e.g. against disseminated TB), it is known to be ineffective with respect to preventing the development, persistence and reactivation of latent TB.
There is an ongoing need to develop improved, more effective vaccines against TB. In particular, there is a need to develop vaccines that provide protection against the development, maintenance and/or reactivation of latent tuberculosis infection.
One protein that has been proposed for use in TB vaccines is the heparin-binding hemagglutinin (HBHA) protein. HBHA is a 22-kDa, methylated, surface-exposed protein that mediates the interaction of the tubercle bacilli with the host, acting as an adhesin for nonphagocytic cells. Methylation of the C-terminal lysine residues is known to affect both the biochemical and immunological properties of the protein, and several experimental findings have implicated HBHA in the process of extrapulmonary dissemination of Mtb (Pethe et al., 2001. Nature 412:190-194.). Temmerman et al. (Nature Medicine 10, 935-941 (2004)) showed that covalent methylation of HBHA is necessary for the elicitation of a protective T cell response in mice challenged with Mtb, and Zannetti et al. showed that purified methylated HBHA is strongly recognized by sera obtained from TB patients compared to controls, whereas unmethylated HBHA is not (Clin Diagn Lab Immunol September 2005 vol. 12 no. 9 1135-1138). In light of these and other studies, it has been proposed that the development of an HBHA-based vaccine may represent an effective strategy to prevent and/or treat TB.
U.S. Pat. No. 7,829,103 to Pethe et al., the complete contents of which is hereby incorporated by reference in entirety, reports immunogenic compositions comprising methylated recombinant HBHA. According to Pethe, the HBHA may be produced by one of two methods: either by 1) producing recombinant non-methylated HBHA protein in a heterologous cell (Escherichia coli or Mycobacterium smegmatis) and then post-translationally methylating the purified recombinant HBHA using a chemical or enzymatic method; or 2) using a recombinant cell to co-express nucleotide sequences encoding HBHA and a mycobacterial methyltransferase. Method 1 involves multiple steps for protein preparation; method 2 involves the use of a heterologous cell that is not administrable as a vaccine. Further, the bacterial strains employed by Pethe were antibiotic resistant, and no discussion of optimizing protein yields is provided. Thus, there remains a need in the art for a recombinant Mtb that is capable of being used as a vaccinogen, and/or for producing sufficient quantities of HBHA to be clinically relevant, both in vitro and in vivo, and/or for producing large quantities of HBHA in a manufacturing setting for later use in clinical applications.